Despite numerous antimicrobial regimens and improved public health, Staphylococcus aureus remains an important pathogen responsible for a number of disease syndromes in humans and animals. This is partly due to the capacity of S. aureus to produce greater than 30 exoproteins many of which have been implicated in the disease process. Definition of most of these proteins as virulence factors has been dependent upon our rather limited ability to mimic the host environment in the laboratory. However, because the staphylococcal infection process remains largely undefined, the investigator hypothesizes that additional undiscovered factors, important to the bacterium, are specifically-expressed only when the organism enters the host environment and therefore, represent new virulence factors. The investigator will investigate this hypothesis by taking the novel approach of combining the method of subtractive hybridization and the capability of growing staphylococci within the host environment to identify genes whose transcription is specifically initiated in vivo. This project will establish S. aureus inside the peritoneal cavity of the rat by means of a diffusion chamber (specific aim 1). Once established, these strains will be used to isolate genes specifically-expressed in vivo by using subtractive hybridization (specific aim 2). Subtracted cDNAs will be cloned and the largest representative of each gene will be identified by cross hybridizing each clone against all other clones. Unique clones will be sequenced, characterized, and assessed for their role in the disease process by replacing the wild- type genes with mutagenized genes (specific aim 3). Strains carrying the mutagenized genes will be inoculated into diffusion chambers and their growth characteristics compared to the parent strains. In addition, these strains will be used in the rat model of cutaneous infection to determine the involvement of each gene as a virulence factor (specific aim 4). It is anticipated that this project will identify genes encoding factors which are important to the pathogenesis of S. aureus and provide the basis for study of these factors as potential candidates for vaccine and antimicrobial drug development.